It takes a perfect arrangement of collagen fibrils and a well-functioning endothelium to make the cornea crystal clear. But that isn’t what keeps it that way, a series of experiments by Boston-area angiogenesis researchers has revealed.

The credit goes to a molecular decoy or “sink” mechanism—an ectopic receptor in the corneal epithelium that latches onto two forms of vascular endothelial growth factor (VEGF), blocking a downstream angiogenic signal that would cloud the stroma with blood vessels.1

The study’s findings suggest the possibility of one day using a synthetic version of the receptor to prevent blindness from diseases like trachoma or from contact lens– induced bacterial infection, or to block vascularization that leads to corneal transplant rejection. Oncologists are expected to investigate whether the receptor’s action will give them a new route to drug or gene-based treatments to choke off a tumor’s vascular supply.

The previously undiscovered role for VEGF receptor-3 (VEGFR-3) explains a wide variety of clinical observations in ophthalmology. For instance, it reveals why:

PRK is more likely to cause corneal haze than LASIK is, and why PRK eyes quiet down after the epithelium regrows. (Removing the epithelium eliminates VEGFR-3.)

Severe infection and inflammation do lead to pathologic angiogenesis. (VEGF molecules greatly outnumber the decoy receptors.)

“The corneal epithelium has this very high concentration of these receptors, and they suck up all the extra VEGF produced in the presence of minor inflammation,” said Reza Dana, MD, MSc, MPH, the senior author of the study. “But when there is a profound infection, the inflammation overwhelms this ‘sink.’” Dr. Dana is a senior scientist at the Schepens Eye Research Institute, associate professor of ophthalmology at Harvard University and head of the cornea service at the Massachusetts Eye and Ear Infirmary.

VEGFR-3 normally is found on endothelial cells lining blood and lymphatic vessels. It binds with two of the five types of VEGF (C and D) to direct embryonic development of lymph vessels, Dr. Dana said. But the receptor’s ectopic and massive expression on corneal epithelial cells was puzzling when Dr. Dana and his team found it several years ago.

The researchers found that, instead of causing lymph vessels to grow in the cornea, the epithelial VEGFR-3 prevents blood vessel formation by immobilizing VEGF-C and -D molecules that otherwise would stimulate further blood vessel growth by binding VEGFR-2—a VEGF receptor that drives pathological angiogenesis and promotes inflammation.

Unbound, the VEGF-C and -D would attract inflammatory cells that in turn would secrete VEGF-A, the isoform most potent for amplifying the angiogenic signal. (This form is the primary target of recent antiangiogenic drugs for age-related macular degeneration and cancer.)

Over the course of three years, the research group ruled out, one by one, alternative explanations for the lack of blood vessels in normal corneas. They showed that VEGF-C and -D bind to VEGFR-3 in the corneal epithelium; that blood vessels grew in de-epithelialized corneas in response to injury or inflammation; that they also grew if the intact corneas were treated with a VEGFR-3 antibody; and that de-epithelialized corneas stayed clear when treated with a synthetic VEGFR-3.

“One of the nice things about this paper is that we really satisfied all the requirements necessary to make a cause-effect relationship stand up. This did not turn out to be merely a correlative study. We actually formally prove that this VEGFR-3 mechanism is there, and what it does,” Dr. Dana said.

This regulatory model might also be at work at the back of the eye, in VEGF-related retinal diseases such as macular degeneration, he said, but further study is needed.

“Whenever people hear ‘VEGF,’ they think it means pathologic angiogenesis. And here we have a part of the eye that is using a VEGF strategy to actually prevent itself from being vascularized,” Dr. Dana said. “It shows you the strong regulatory systems the body has to prevent pathology.”

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1 Proc Natl Acad Sci USA 2006;103(30):11405–11410.

Retina Update

Antioxidants Effective in RP Mice

The gradual death of cone photoreceptors in retinitis pigmentosa patients might be prevented by high doses of injected antioxidants, according to a study by researchers at Johns Hopkins University.1

The preventive effect has only been shown in mice, and a reliable delivery method for protection against oxidative damage would be necessary before this approach could reach the clinic, notes the paper by a team led by Peter A. Campochiaro, MD, professor of ophthalmology and neuroscience. But this discovery showing that after rods die, cones die from oxidative damage has “important clinical implications,” the researchers add.

“It provides a therapeutic target that may apply to all (or most) RP patients. The enormous genetic heterogeneity among the diseases that constitute RP is a problem for the development of treatments that deal with primary genetic defects . . . ,” they write. “If, regardless of the genetic defect that leads to rod-cell death, the same treatment can be used to prolong cone survival and function, the impact would be enormous. As long as cones survive, useful vision is possible.”

The study investigating the effects of antioxidants on cones in a model of RP was inspired by an unexpected finding several years ago. Adult mice were placed in a high-oxygen environment to study the effect on retinal vessels, and it was found that after several weeks the photoreceptor cells died. A review of the literature showed that this same observation had been made in 1955 by neurophysiologist Werner K. Noell, PhD, of the State University of New York at Buffalo.2 He was renowned for his pioneering work on light damage to the retina. This is a very important observation because it indicates that photoreceptors are exquisitely sensitive to oxidative damage.

Rod photoreceptors are more numerous than cones and are the major oxygen consumers in the retina. After rods die, the oxygen level in the outer retina is substantially elevated just like when mice breathe high levels of oxygen. This led to the hypothesis that the gradual death of cones, which occurs after rods die in patients or animals with RP, is due to oxidative damage.

To test the hypothesis the Campochiaro group subjected mice with RP to a mixture of antioxidants including vitamin C, vitamin E, alpha-lipoic acid and MnTBAP, a metalloporphyrin that mimics a superoxide dismutase.

At postnatal day 35, mice receiving daily injections of the mixture had about twice as many cones as mice that received vehicle alone, indicating that protection from oxidative damage reduced cone cell death in this mouse model of RP.

This proves the hypothesis and suggests that future research to focus on finding ways to maximize protection from oxidative damage could lead to treatments that provide benefit in RP patients regardless of the underlying genetic defect.

A continuing effort to plumb data from the Blue Mountains Eye Study using the retina as a window on the systemic microcirculation has yielded a new cardiovascular risk marker. But it would be visible only with retinal photography.

The study found that evaluation of the relative size of arterioles and venules showed that there was one patient group—women from age 49 through 75—for which size of these vessels at baseline could indicate a 50 percent higher risk of dying of coronary heart disease in the subsequent nine years.1

A smaller arteriole-to-venule ratio (AVR) and narrower arterioles were two measures that the study found correlated with CHD-related death.

But the size differences in these small vessels are very subtle, warned lead study author Jie Jin Wang, PhD, a senior research fellow at the Centre for Vision Research in Sydney, Australia. “Funduscopic examination by the general physician generally would not be able to detect these subtle retinal vessel changes, with reasonable reliability,” she said.

It is also too early to know how, or if, this would affect ophthalmic practice. “Ophthalmologists should be made aware of this association, but the findings are not yet ready to be applied directly to clinical practice,” Dr. Wang said.

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1 Wang, J. J. et al. Heart 2006 Jul. 13 [Epub ahead of print].

Research Report

How to Assassinate Angiogenesis

Instead of chasing down growth factors like VEGF, why not inactivate the downstream genetic machinery that VEGF mobilizes?

That central question underlies a different approach to controlling angiogenesis —an approach that is yielding results that appear applicable to diseases as diverse as ocular neovascularization, rheumatoid arthritis and cancer.

An Australian research group reported on using “molecular assassins” to silence a gene transcription factor involved in neovascularization, increased vessel permeability and inflammation.1 The molecules were small interfering RNA (siRNA) oligonucleotides, or complementary pieces of RNA, and “DNAzymes,” the catalytic DNA equivalent of a ribozyme.

“Transcription factors are ideal targets because these genes in turn control multiple other disease-causing genes. It’s like looking at a chain of dominoes, and you’re trying to keep them from falling by targeting one of the early dominoes,” said Levon Khachigian, BSc, PhD, DSc, a professor of hematology at the University of New South Wales in Sydney.

In experiments in a rat model of retinopathy of prematurity, Dr. Khachigian’s group reported successfully disabling the transcription factor c-Jun. Normally dormant in adult cells, c-Jun becomes active in the presence of VEGF, setting off a chain of other genes involved in vascular proliferation and inflammation.

Dr. Khachigian said that c-Jun also appears in inflammatory diseases such as rheumatoid arthritis.

“With our molecular assassins, we have successfully been able to prevent neovascularization using both DNAzymes and siRNA,” he said.

If the c-Jun blockade strategy proves effective in humans, Dr. Khachigian said it would be logical to pair c-Jun-blocking therapy with existing or future pharmacologic agents that block VEGF itself. One molecule would remove VEGF from the target cells, and the other would silence gene transcription factors that the VEGF had already activated. The University of New South Wales is looking for commercial partners for clinical studies of the c-Jun blockade strategy.

“We’re not proposing a chronic treatment. We’re talking about acute treatment,” Dr. Khachigian said, adding, “Just a single bolus administration of the agent by intravitreal injection prevents the appearance of neovascularization at the back of the eye.”